This invention relates to a method whereby living cells modified by a biological substance, introduced into them by ballistic transfer, are efficiently separated from remaining unmodified cells.
Many methods of modern cell biology require the transfer of matter, mainly nucleic acids, into living cells (hereafter referred to as transfection). Traditionally, this transfer of matter has been important to both the fields of biological and medical research. Recent progress, however, in the understanding of the body""s functions as regarded to molecular mechanisms has led to the idea of treating human desease by using molecular approaches (colloquially referred to as xe2x80x9cgene therapyxe2x80x9d). Many of the biological methods suggested in this approach require the transfection of somatic cells. A number of techniques have been developed to achieve this aim: microinjection; electroporation; transfection by viral vectors or liposomes; and direct bombardment of cells with particles (xe2x80x9cgene gunxe2x80x9d). For a review on methods see Methods in Enzymology 217, (1993), pp. 461-655, (Academic Press, San Diego, Calif.).
Apart from microinjection, in which a single cell is injected directly with the transfecting matter, these methods suffer from a rather low and unreliable efficiency, efficiency being measured as percentage of successfully transfected cells out of total of treated cells. Microinjection""s efficiency is very high; however, the number of treated cells is generally too low for this technique to be clinically valuable.
If the object of the transfection is to insert genetic information into the cell, then successful transfection requires the passage of the transfecting nucleic acid not only into the cell cytoplasm, but into the nucleus. The nuclear membrane is a barrier more difficult to cross than the cytoplasmic membrane. Many of the cells transfected by means of electroporation or lipofection that have incorporated the transfecting matter into their cytoplasm, will not express any genetic message transferred into them. For expression of any genetic message to happen, the genetic message has to pass into the nucleus. The transfection of cells with DNA by electroporation is most likely successful only when it happens during cell division, because the division process momentarily renders the nucleus permeable for the transfecting DNA.
In contrast, the ballistic transfection method achieves transport into the nucleus by the kinetic energy of the passing particle. The probability of nuclear passage of the microcarrier particle is governed by the ratio of nucleus diameter to cell diameter, which for many cells, is very favourable for nuclear passage. Thus, it can be expected that any clinical approach to transfection of cells with DNA would increase efficiency, employing the ballistic transfection method.
A current estimate of the number of transfected cells needed in a clinical protocol is in the order of 107-108 cells. For the reasons given above, we believe that of the transfection methods mentioned, the ballistic transfer, i.e. directly bombarding cells with particles that carry the transfecting matter into the cells, has the greatest potential to achieve this aim.
Various embodiments of the idea of bombarding cells in order to achieve transfection have been published. They differ in the propulsion of the particles, the nature of the particles and various other aspects. A number of patents have been filed describing these embodiments (see: Jones, Frey, Gleason, Chee, Slightom: Gas driven microprojectile accelerator and method of use U.S. Pat. No. 5,066,587; Jones, Frey, Gleason, Chee, Slightom: Gas driven microprojectile accelerator WO 9111526, U.S. Pat. No. 471,216; Sanford, Wolf, Allen: Apparatus for delivering substances into cells and tissues in a non-lethal manner EP 0 331 855; Tome: improved particle gun EP 0 397 413; Brill, McCabe, Yang: Particle-mediated transformation of animal somatic cells WO 91/00359; Mets: Aerosol beam injector WO 91/00915; WO 91/02071; Johnston, Williams, Sanford, McElligott: Particle-mediated transformation of animal tissue cells WO 91/07487; Bruner, deVit, Johnston, Sanford: Improved method and apparatus for introducing biological substances into living cells WO 91/18991; Bellhouse, Sarphie: Ballistic apparatus. WO 9204439, GB 9018892.1). However, only one embodiment to our knowledge, is commercially manufactured. This embodyment is the xe2x80x9cBiolisticxe2x80x9d apparatus invented by John C Sanford and manufactured under licence from Cornell University and DuPont by Bio-Rad (Hercules, Calif.). The propulsion of the microcarriers is achieved by adsorbing the microcarriers to a macrocarrier polymer sheet, which is accelerated towards the cells by a cold gas shock wave. After retaining the macrocarrier, the microcarrier sheaf continues towards the target cell layer, eventually impacting and unloading the adsorbed transfecting matter into the cells.
The method of ballistic transfection implies that only a (sometimes large) fraction of the target cells is transfected successfully. The microcarrier sheaf is rarely homogeneous, and has to be of sufficiently small density in order not to kill too many of the target cells, which invariably suffer from stress exerted on them by both the shock wave and the impacting microprojectiles. A balance must be found between a high survival rate and a high transfection rate, which leaves part of the target cells untransfected.
In many of the plausible clinical uses, a separation of transfected cells from untransfected cells is desirable, if not strictly required. Ex vivo transfection of tumor cells for cancer gene therapy is only one example. The current state of the art employs time-consuming separation protocols based on expression of markers. The genetic information for these markers is introduced into the cell with the transfecting DNA. This procedure requires cell culture of the transfected cells ex vivo for a prolonged period of time, raising the risk of both contamination and alteration of cell characteristics. A method enabling a quick, simple separation of transfected cells would clearly be of great value.
Magnetic separation techniques have been in use in biology for years. These methods primarily employ paraferromagnetic beads, the size of which ranges in micrometers. Paramagnetic particles of such size retain some residual magnetic orientation after removal of an external magnetic field, leading to aggregation in solution. Recently, a new separation technique has been introduced (Miltenyi: Methods and materials for high gradient magnetic separation of biological materials WO 90/07380; DE 3720 844) that is based upon the coupling of biological material onto submicroscopic-size magnetic particles. These particles have the property of being xe2x80x9csuper-paramagnetic,xe2x80x9d meaning their magnetic core is smaller than the the size of a Weiss domain: the area of similar magnetic dipole orientation within a paraferromagnetic solid. In the absence of an external magnetic field, these particles do not retain any macroscopic magnetic orientation; thus do not attract each other, making them ideal for suspension in fluids. Their retainment requires a very strong xe2x80x9chigh gradientxe2x80x9d magnetic field, since the magnetic force exerted on the particle is small due to its minimal size. A typical separation apparatus employs a mesh of iron wool embedded in polymer, through which the suspension is passed. In the presence of a strong magnetic field, the local field inside the wool mesh is strong enough to retain the particles. After removal of the external field, the particles can be washed out.
The cytograms show the results of fluorescence measurements. The abscissa represents fluorescence in log scale, the ordinate cell number. U represents unsorted cells after ballistic transfer; M and N represent the magnetic and nonmagnetic fractions, respectively.
The invention refers to a method by which cells that have been transfected by ballistic transfer methods (xe2x80x9cgene gunxe2x80x9d) can be separated from untransfected cells. It employs co-adsorption of nanometer-sized xe2x80x9csuperparamagneticxe2x80x9d particles and the transfecting matter onto the microcarriers. The microcarriers are accelerated towards the cells. They hit the cells and unload the magnetic particles into the cells, along with the transfecting matter. The magnetic particles render the cells susceptible to magnetic field forces. After the ballistic transfer procedure, the cells are suspended in medium and passed through a separation column within a strong magnetic field. Transfected cells are retained by the incorporated magnetic particles, whereas non-transfected cells are washed through. After removal of the external field, separation yields of over 90% successfully transfected cells are achieved.
The claimed method allows the easy separation of cells that have been hit by one or more microcarriers. After such contact, the cells are presumably loaded with the transported matter. In our experiments, we used fluorescence-tagged oligodesoxynucleotides to transfect cells from human cell culture lines by a procedure that employed the xe2x80x9cBiolisticxe2x80x9d-transfer technology marketed by xe2x80x9cBio-Rad.xe2x80x9d We found that after transfection, more than 95% of the surviving cells separated by the invention described here were marked with fluorescence, and, therefore, successfully transfected. This fluorescence reading constitutes a substantial increase compared to the same experiment conducted without separation, typically yielding a fluorescence-positive cell count of 40%.
It is conceivable to use other microcarriers than the 1.6 micron gold particles employed in the examples set forth within this disclosure. The separation technique is not confined to the apparatus, particles, cells or propulsion means of the microcarriers used here. More specifically, it is conceivable to use paramagnetic particles as both microcarriers and magnetic means of achieving the separation.
The claimed separation method is especially useful in the field of cancer gene therapy: An important approach to treating tumor patients is the immunization of the patient against the tumor cells. This is achieved by excision of tumor tissue, and transfection of excised tumor cells with plasmids carrying expressable genes that encode proteins. The encoded proteins, when expressed by the tumor cell, direct the immune system response against the cell. Among the proteins suggested in this context are cytokines interleukin 2 and 7, major histocompatibility complex proteins, growth factors and others.
After transfection, cells are incubated under conditions suited to trigger expression of the transfected gene for 24 h to 48 h. The cells are then irradiated to render them incapable of cell division, and given back into the patient subcutaneously in most cases. Alternatively, intravenous transfusion may be used if haemodynamic or other mechanisms of distribution and/or homing are intended. The irradiated cells continue to express their genetic programm, part of which is the transfected gene for the immunostimulant protein. The body""s immune system responds to the transfected cells by mounting an immune response against the transfected tumor cells. The immune system, thus taught how to recognize and fight the tumor cells, continues to attack not only the transfected tumor cells but also the untransfected cells of tumor tissue that had not been excised and possible metastases.
For this approach, it is important to treat the patient with successfully transfected cells only. The achievable immune response is strongly dependent on the concentration of the expressed gene product. Any significant contamination of untransfected cells not only reduces the concentration of the gene product, but will tend to overgrow transfected cells during the 24 h to 48 h incubation period. Thereby, the concentration of the gene product of the transfected gene becomes exponentially diluted and clearly reduces the desired immune response. The present invention is the only separation method to our knowledge that enables the neccessary separation yields for gene therapy.
In the case of melanoma, leukemia and few other tumor tissues it is possible to obtain primary cell cultures of tumor cells relatively easy. These primary cell cultures are transfected preferably by ballistic transfer, because the present separation method provides for easy separation of transfected cells.
However, the advantage of the combination of ballistic transfer and magnetic separation becomes even more obvious in the case of cancers as mamma carcinoma, colon carcinoma and rectum carcinoma. These cancers are among the epidemiologically most relevant malignant deseases. Their dependancy of growth on tissue environment makes it impossible to establish a reliable primary cell culture and transfect them subsequently. If a few cells adhere to the petri dish and start to proliferate, they bear little immunological resemblance to the original tumor cells. If the approach outlined above is to be used to induce an immune response of the patient against the tumor, solid tumor tissue has to be treated. Within the relatively intact microenvironment of a slice of excised tumor tissue, the cells on the surface of the slice are a population representative of the tumor, as seen by the body""s immune system.
Our approach is to transfect slices of 500 xcexcm thickness, which corresponds to about 10 layers of cells. We estimate that by xe2x80x9cshootingxe2x80x9d at both sides of a tumor slice, we reach 20% of the cells in the slice, of which 10% to 40% are succesfully transfected, resulting in a net efficiency of 2% to 8% of treated tumor slice. It is obvious that a separation of successfully transfected cells is of paramount importance to this approach to treating these epidemiologically, and thus economically, most important cancers.